Significant research efforts have been directed towards developing systems for enhancing the intracellular delivery of phosphate-substituted compounds, such as biologically active peptides and nucleotides, inasmuch as the high negative charge of these compounds leads to very low cellular permeability. For example, there is much interest in the intracellular delivery of tyrosine phosphate peptides and peptidomimetics for use in blocking the uncontrolled proliferation of tumor cells. In this regard, protein tyrosine kinases are important regulators of cell cycle progression, and represent attractive targets for the rational design of novel anticancer agents. Protein tyrosine kinases are activated upon phosphorylation of specific tyrosine residues of the kinases and the phosphotyrosine residue along with the three adjacent amino acids (pY-E-E-I) serve as a binding site for Src homology 2 (SH2) domains of intracellular signaling proteins. Interaction between an SH2 domain of an intracellular signaling molecule and an activated protein tyrosine kinase is essential for effective cell cycle progression. Thus, molecules that mimic the phosphotyrosine moiety of protein tyrosine kinases and block the binding of protein tyrosine kinases to SH2 domains represent promising antiproliferative agents for the treatment of cancers.
The phosphate dianion on tyrosine-phosphorylated protein tyrosine kinases is critical to the kinase-SH2 domain interaction. The crystal structures of a number of ligand-SH2 domains have been reported, the interactions have been modeled, and many different inhibitors have been synthesized. In all of these phosphotyrosine peptides and peptidomimetic inhibitors, the phosphotyrosine residue is involved in a complex network of hydrogen bonding and charge-charge interactions that require extensive interaction of the SH2 domain with the phosphate dianion of the tyrosine phosphate moiety. The importance of this dianionic interaction is illustrated by the fact that substitution of the phosphate group with a wide variety of other anionic and neutral hydrogen-bonding substituents leads to a significant loss in binding affinity. Thus, the intracellular delivery of tyrosine phosphate peptides and peptidomimetics where the phosphate is in the form of the dianion is critical.
Nucleotide analogs represent another class of promising agents for use in treating disease states caused by uncontrolled viral or cellular replication. Like tyrosine phosphate peptidomimetics, the phosphate dianion of nucleotide analogs is critical to the capacity of these compounds to block viral or cellular replication. The most widely used approach for the use of nucleotide analogs as therapeutic agents is to deliver the nucleoside analog as a prodrug across the cell membrane and to rely on the cellular machinery to link a phosphate group to the purine or pyrimidine base to form the corresponding nucleotide analog. However, many nucleosides with therapeutic promise are not converted to the corresponding nucleotide by existing cellular pathways. Furthermore, tumor cells or virus-infected cells may become resistant to treatment by down-regulating the activity of nucleoside kinases, and, for many previously developed nucleotide prodrugs, the intracellular activation process is so inefficient that effective nucleotide concentrations do not accumulate within the cell.
The general strategy for the intracellular delivery of phosphate-containing compounds utilized in the present invention is outlined in Scheme 1:
Briefly, the phosphate-substituted compound to be delivered intracellularly is synthesized (R with attached phosphate in scheme 1 above) and is linked to a delivery group and a masking group. The masking group consists of a haloalkylamine moiety that is hydrolyzed intracellularly to produce an intermediate that is stable as long as the delivery group remains intact, but is released upon intracellular activation of the delivery group. The delivery group may be a group, such as a nitrofuryl group or a perhydrooxazine, that is subject to intracellular hydrolysis to provide an intermediate which undergoes cyclization and P—N bond cleavage (i.e., spontaneous hydrolysis) in the intracellular environment releasing the desired phosphate-containing compound.
Systems for the intracellular delivery of alkylating agents and nucleotides that employ chemistry related to that described in scheme 1 have been developed previously. However, previous methods resulted in only about 50% intracellular conversion to the desired nucleotide with the other 50% of the prodrug being “lost” as a biologically inactive solvolysis product. The present invention is directed to a newly discovered modification of this chemistry that extends its applicability both to nucleotide analogs and to the phenol phosphate group that is the essential constituent of tyrosine phosphate peptides and peptidomimetics. The invention is based on the discovery that changing the structure of the masking group from a haloethyl to a halobutyl (or halopentyl) group results in the rapid and quantitative intracellular conversion (i.e., about 100% conversion) to the desired nucleotide or phosphate-containing peptide. The new chemistry of the invention is applicable to nucleotide analogs, phosphotyrosine peptides and peptidomimetics, and to a wide variety of other pharmaceutically significant compounds containing phosphate moieties.
The development of protected phosphotyrosine precursor phosphoramidates in accordance with this invention enables facile synthesis of phosphotyrosine peptides and peptidomimetics. Thus, the invention is also directed to protected phosphotyrosine precursors and related protected peptides, for example, with N-BOC and fluorenylmethoxycarbonyl groups.
In one embodiment of this invention there is provided a phosphoramidate prodrug compound wherein a biologically active compound having hydroxy functionality is covalently linked through that hydroxy functionality to form a biologically labile phosphoramidate which is converted intracellularly to the corresponding drug phosphate. The biologically labile phosphoramidate group enables more efficient intracellular delivery of the drug substance and serves as a means for achieving biologically significant intracellular concentrations of the drug substance in the form of its corresponding phosphate ester. In another embodiment the biologically active compound has amino functionality, and it is linked through that amino functionality to form a biologically labile phosphoramidate which is converted intracellularly to the phosphoramide (−OPO2NH-Drug).
In another embodiment this invention provides an intermediate halophosphoramidite of the formulaRrCH2OP(halo)NR(CH2)nXwherein n is 4 or 5, R is lower alkyl or —(CH2)nX, X is an electrophilic group capable of being nucleophilically displaced from the carbon atom to which it is bound and the group RrCH2— is a biologically labile ester forming group, more particularly, an ester forming group that is readily hydrolyzed intracellularly. This compound can be used as an intermediate reagent for preparing the above-described phosphoramidate prodrug compounds using a method embodiment of this invention wherein the biologically active compound (Drug-OH) is reacted with that halophosphoramidite intermediate to form the corresponding drug reacted intermediateRrCH2OP(O-Drug)NR(CH2)nXwhich is then oxidized to form the phosphoramidate prodrug. The method can be used to form phosphoramidite precursors of intracellular drug phosphates starting with hydroxy functional amino acids having carboxyl and amino functional groups protected with standard protecting groups or from biologically active peptides or peptidomimetics or nucleotide analogs. The phosphoramidate prodrugs in accordance with this invention are converted to the corresponding phosphates following hydrolysis of the RrCH2— ester forming group. Subsequent hydrolysis of the cyclized 5- or 6-membered zwiterionic intermediate (formed by cyclization of the group —N(CH2)nX) provides the corresponding phosphate as the exclusive phosphorous-containing product. Analogous synthetic procedures can be carried out with active compounds having amino functionality represented generally herein as Drug NH2.